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Design and Engineering Next-Generation Nanopore Devices for Bioplymer Analysis

Periodic Reporting for period 4 - DeE-Nano (Design and Engineering Next-Generation Nanopore Devices for Bioplymer Analysis)

Reporting period: 2022-01-01 to 2022-06-30

The main objective of this work is to design and engineer nanopores that will be used in biopolymer analysis, with the ultimate goal of sequencing single proteins. The ability of sequencing proteins at the single-molecule level is important in industry and base science. Proteins are biomarkers linked to disease, hence devices capable of identify proteins are important in medical diagnostics. In addition, while all cells have the same DNA, the expression of proteins is unique. Hence, if we want to understand how a cell works, we need to understand how proteins are made and modified. To date there is no single-molecule technique to sequence proteins. Single-molecule sequencing is important because proteins exist in cells in highly variable and heterogeneous mixtures. And only single-molecule techniques can address this issues.

During the duration of the proposal, we have taken two approaches. One was to engineer nanopores for the de novo identification of proteins. In this respect, we have built a biological nanopores with an embedded peptidase. An unfoldase would then select, unfold and deliver proteins to a nanopore-peptidase. The latter would then identify the individual fragmented peptides. In the next step, we have shown that if the peptides are cleaved in a well-defined manner (e.g. after positively charged residues as for the digestion with trypsin), proteins can be identified. Finally, we showed that the sequential identification of peptides at the single-molecule would lead to the identification of 98% of proteins in the proteome.
The nanopore was also able to transport intact proteins across the nanopore. Hence, we also demonstrated that our system is capable of characterising single proteins during their intact transport across the nanopore. In this 'mode' the peptise activity is removed and the identification amino acid by amino acid might be possible.

In a second single-molecule identification approach, we discovered that engineered nanopores could identify very selectively hemoglobin in blood. Substitution of a single amino acid, a condition that occurs in hemophilic patients, could also be detected. This approach is amenable of real-time and single-molecule identification in complex biological samples.
In the final report year we have focussed on the identification of peptides using nanopores. We have found that the engineering of a beta-barrel nanopore with aromatic residues allows the detection of a wide variety of polypeptides. In addition, with the engineered nanopores we have shown that a variety of isomeric peptides can be distinguished. We have also engineered the artificial nanopore to work with a different unfoldase. Unfortunately, further engineering of the nanopore-peptidase revealed unsuccessful. New methodologies will be developed to couple paptidases with nanopores. On the other hand, we have found that polypeptides can be unfolded and translocated under the control of an unfoldase
We have described how to design a nanopore that contains a bespoke beta barrel region and it includes a soluble protein of choice. In addition, we have genetically fused a peptidase to the nanopore and built a nanopore with an emergent enzymatic property. This was never described before in the literature. The ability of adding a beta barrel into a solubile protein and insert into a lipid bilayer will allow the use of a range of constructs for the sequencing of protiens.
We also have shown that nanopore currents can be used to identify the volume of peptides. This is an unexpected and important discovery, as it open to the making devices that can compete with mass spectrometry devices. Using this discovery, we described a method for using nanopores to identify proteins.
Interestingly, we have e found that nanopores can identify enantiomer and diasterosomer differences in polypeptides. Finally, we have shown that nanopores can also be used to identify proteins and their modifications directly in complex biological samples.
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